Oxidative stress generated during cerebral ischemia and reperfusion is a critical event leading to damage of the neurovascular unit that causes blood-brain barrier disruption, with secondary vasogenic edema and hemorrhagic transformation of infarcted brain tissue, restricting the benefit of tissue reperfusion with thrombolytic agents. However, little is known about the cellular and molecular mechanisms and signaling pathways that underlie oxidative stress in the damage of the neurovascular unit that consists mainly of endothelial cells, astrocytes, and neurons. Our working hypothesis is that NADPH oxidase, a pro-oxidant enzyme that localizes in all the cells of the neurovascular unit, initiates oxidative stress and downstream signaling that cause neurovascular damage and intracerebral hemorrhage in the stroke brain. This Program consists of three interactive projects and two supporting cores. Project 1 is to investigate the role of endothelial NADPH oxidase in oxidative stress, endothelial cell damage, and intracerebral hemorrhage. Project 2 will elucidate the role of the interplay between hyperglycemia and oxidative stress generated by NADPH oxidase in ischemic neuronal injury. Project 3 will elucidate astrocytic HSP70 and NADPH oxidase in endothelial protection and injury in the ischemic brain. We believe these are novel studies that will provide insights into the oxidative mechanisms in neurovascular dysfunction after cerebral ischemia. The long-term goal of these studies is to develop therapeutic interventions that target neurovascular function for stroke patients. Lay statement: Most strokes are caused by occlusion of the cerebral artery by a thrombus (ischemic stroke). Early restoration of cerebral blood flow (reperfusion) with a thrombolytic agent or spontaneous reperfusion can salvage brain tissue, but can also have a deleterious effect by generating reactive oxygen species by NADPH oxidase. The latter can further damage neurons and vessels. This Program aims to elucidate the role of NADPH-oxidase in reperfusion injury and to provide strategies to prevent or to ameliorate brain damage in patients with acute stroke. Project 1 Title: Neurovascular Dysfunction, BBB Disruption and Oxidative Stress in Ischemic Brain PI: Pak Chan, PhD DESCRIPTION (provided by applicant): Oxidative stress generated during cerebral ischemia and reperfusion is a critical event leading to blood brain barrier (BBB) disruption, with secondary vasogenic edema and hemorrhagic transformation of infarcted brain tissue, restricting the benefit of tissue reperfusion with thrombolytic agents. We have demonstrated that mild cerebral ischemia (30 minutes) in mice deficient in manganese-superoxide dismutase (SOD2-/+) caused delayed (>24 hours) BBB breakdown associated with activation of matrix metalloproteinase (MMP), inflammation, and high brain hemorrhage rates. Our preliminary studies have further shown that overexpression of endothelial NADPH oxidase is associated with the formation of hemorrhagic transformation and intracerebral hemorrhage (ICH) in the SOD2 -/+ mice, and that inhibition of NADPH oxidase significantly reduces ICH and infarct volume. We now hypothesize that activation of endothelial NADPH oxidase and expression of extracellular signal-regulated kinase (ERK) 1/2 signaling cause neurovascular dysfunction, BBB disruption, and endothelial cell death by activation of MMP-9. Our aim is to test this hypothesis using this newly developed model of SOD2-/+ mice with delayed BBB disruption. We believe these are novel studies that will provide insights into therapeutic opportunities to minimize oxidative stress-associated hemorrhagic transformation in patients who suffer acute ischemic stroke.